Changes Of Amylase And Sucrose Activity During Lily Bulb Dormancy And Cloning Of ARF Gene

Lily is a famous bulbous flower,with high ornamental,edible and medicinal value.Lily bulbs in the development of mature will gradually enter the dormant state,no breaking of dormancy lily bulbs will appear low germination rate and blind flower phenomenon after planting.The expression of ARF gene affects the activity of sucrose phosphate synthase,participates in the synthesis of auxin and promotes the development of plants.ArfGTPase activates proteins that affect the polar transport of auxin.In this study,we analyzed the changes of amylase and sucrase activity and the expression of ARF gene in the four important periods during the dormancy release process,and cloned the ArpGTPase activator protein LpAGD14 gene,and carried out bioinformatics analysis and plant expression Vector construction,lay the foundation for subsequent functional verification.The main results of this study are as follows:1.The activity of amylase,starch phosphorylase,sucrose synthase and sucrose phosphorylase in four stages of dormancy,germination,bud growth and budding period showed that the activity of starch metabolism related enzymes Is low,the lily bulb is higher in this period,the metabolic activity is slow,the level of soluble carbohydrate is low,the rate of starch conversion to soluble sugar is low,leading to the initial sucrose phosphate synthase activity increased,promote sucrose phosphate synthesis of sucrose.During the period from the beginning of the dormancy release to the complete release,the metabolic activity was rapid,the related enzyme activity increased,the soluble sugar consumption was fast,and the activity of sucrose synthase was the highest.The outer scale amylase was generally higher than the inner scale,indicating that the metabolic activity of the inner scale was lower than that of the outer scale.2.The results showed that these genes were mainly involved in the biological processes such as amino acid metabolism,coenzyme synthesis,glucose metabolism,signal transduction and so on.The results showed that these genes were mainly involved in the process of amino acid metabolism.The 15 genes were up-regulated and the specific primers were designed.The real-time quantitative experiments were carried out to verify the expression of these genes at different times.The results showed that the expression level of all the genes increased in four periods,the highest level of dormancy at the beginning of storage,the lowest gene expression and the highest expression of related genes when the dormancy was completely released,indicating that these genes were up-regulated genes The significance of the study.3.The LpAGD14 gene cloned from the Lilium pumilum cDNA belongs to the ARF gene family and has a typical conserved domain of ArfGAP.It is predicted by protein function that it has the same activity as GTP activator protein AGD14 of various plants such as Elaeis guineensis,Musa acuminata subsp,and Asparagus officinalis.High homology,suggesting that the protein belongs to the Arf class GTPase activator protein,can regulate auxin influx,vesicular transport and root development,in order to further study the function of ARF gene lay the foundation.4.The plant expression vector pBI121-LpAGD14-GFP was constructed and the GFP fusion protein was used to express the GFP fusion protein into the onion epidermal cells.The green fluorescence was found in the cell membrane and nucleus of the onion epidermal cells,indicating that the LpAGD14 gene was expressed in the cell membrane expression.The plant expression vector pCXSN-LpAGD14 was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens.The results showed that the foreign gene was successfully transferred into Arabidopsis thaliana Genome.